Diagnosis of Pulmonary Tuberculosis and Detection of Resistance to Rifampin and Isoniazid through Direct Molecular Methods in Stool Samples

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1 616 Available online at Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 Diagnosis of Pulmonary Tuberculosis and Detection of Resistance to Rifampin and Isoniazid through Direct Molecular Methods in Stool Samples Sulaiman Ali Al Yousef 1, Khalid Abdullah Ali AbdelRahim 2,3, Asmaa O. Ahmed 4, Khalid Salmeen Almaary 2, and Ahmed Mohamed Ali Mohamed 1 1 Department of Medical Laboratory Technology, College of Applied Medical Science, University of Hafr Albatin, Hafr Al Batin, 2 Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia, 3 Botany Department, Faculty of Science, Sohag University, Sohag, and 4 Department of Clinical Pathology, Clinical Microbiology Unit, Faculty of Medicine, Assiut University Hospital, Egypt Abstract. Background. Tuberculosis (TB) is an infectious disease that is caused by Mycobacterium tuberculosis (M.tb). TB has high morbidity and mortality around the world. Objective. To evaluate molecularbased methods performed directly on stool samples for the diagnosis of pulmonary tuberculosis (PTB) and to determine the susceptibility to Rifampicin (RMP) and Isoniazid (INH). Study design. This is a descriptive study evaluating the performance of the PCR-based method for direct PTB diagnosis and to determine the susceptibility of RMP and INH using stool samples from PTB patients. The study was conducted between March 2011 and March Methods. Three stool samples and three sputum samples (n=300 stool and 300 sputum) were collected from 100 PTB patients (75 pretreatment and 25 follow up). Stool samples (n=60) were also collected from 20 healthy individuals to serve as controls. DNA was extracted from stool samples using Chelex PTB was diagnosed using a Genekam kit. RMP and INH susceptibility testing was performed using the Genotype MTBDRplus assay. The sputum Lowenstein Jensen (LJ) culture and agar proportion method (PM) of drug susceptibility testing for INH and RMP were considered to be the gold standard methods for comparing the results of the molecular methods. Results. The Genekam kit showed 100% sensitivity and 95.24% specificity for diagnosing new patients and showed 100% sensitivity and 80% specificity for follow-up patients. The Genotype MTBDR-plus assay showed 86.4% and 100% sensitivity and 98.1% and 97.8% specificity for determining INH and RMP sensitivity, respectively, in newly diagnosed patients and 85.7% and 94.4% sensitivity and specificity, respectively, for both INH and RMP in follow-up patients. Conclusion. Molecular-based methods are promising techniques for the diagnosis and susceptibility testing of PTB when the ease of sample collection and the speed of diagnosis are taken into consideration. However, they are not as useful for assessing follow-up patients. Key words: Tuberculosis, MTDR-plus, Genekam, MDR-TB, Stool, Egypt. Introduction TB is a worldwide pandemic and a serious cause of illness and death. In 2013, an estimated 9.0 million people developed TB, and 1.5 million died from this disease [1]. The diagnosis of PTB currently depends on the detection of Mycobacterium tuberculosis complex (MTC) organisms in the sputum [2]. Some Address correspondence to Ali AbdelRahim, Botany and Microbiology Department, College of Science, King Saud University P.O. Box 2455, Riyadh 11451, Saudi Arabia; e mail: kabdelraheem@ksu.edu. sa, khalidfp7@gmail.com patients, however, are unable to produce sputum, including children [3], immune-compromised patients, and patients with neurological impairment [4]. In these situations, alternative specimens are obtained by invasive procedures include nasopharyngeal aspirates [5], gastric aspirates [6], respiratory tract secretions is obtained by bronchoscopy [7], and intestinal fluid is obtained by the string test [8]. These invasive procedures are uncomfortable for the patient and are potentially harmful. Because swallowed MTC organisms can be detected in the gastric fluid and have been recently demonstrated to resist acid [9]. It is possible that MTC /16/ by the Association of Clinical Scientists, Inc.

2 Direct Molecular Methods for Diagnosis of Pulmonary Tuberculosis 617 Table 1. Age and sex distribution of the patients and controls (healthy individuals). No subjects included in our study were HIV positive. Control No. Patients No. Group years years years Male 7 46 Female organisms may survive the enteric tract and be eliminated in stool. However, few systematic evaluations of the utility of stool specimens for the diagnosis of PTB have been reported [10]. Due to the development of drug-resistant M.tb isolates, rapid and reliable methods for the diagnosis and drug susceptibility testing (DST) of TB are urgently needed. The conventional methods for DST of M.tb include PM on LJ medium or Middlebrook 7H10 agar [11] and MGIT (BACTEC 960 or 320) [12]. They require either several weeks to yield results or depend heavily on mechanized equipment that uses radioactive compounds. In contrast, the sputum PCR method can be performed in one day and has a sensitivity of 60 to 100% [13-15]. Furthermore, PCR allows for direct determination of RMP resistance [16,17], which is particularly important because it is a marker of multidrug-resistant strains of M.tb (MDR-TB)[18,19] and a strong predictor of treatment outcome to detect RMP and INH resistance in M.tb isolates., A DNA strip assay was developed (Genotype MTBDRplus; HainLifescience, Nehren, Germany) based on a multiplex PCR in combination with reverse hybridization to identify either wild-type sequences or specific mutations. The aim of this study was to evaluate PCR-based methods for the detection and susceptibility testing of RMP- and INH-resistant MDR-TB in stool samples from patients with PTB. Genekam TB kits for the detection of M.tb and the Genotype MTBDR-plus assay for RMP and INH susceptibility testing. Materials and Methods Biosafety. The following procedures were conducted under biosafety level 2 standards at the Tuberculosis Reference Lab, Ministry of Health Central Labs, Cairo, Egypt, from March 2011 until March Study design. Egypt, as a country, is ranked with a middle-low level of TB incidence according to the WHO classification. It is estimated that 11 case per 100,000 population develop active pulmonary smear-positive TB out of a total of 24 cases per 100,000 population who develop all types of TB annually. Most cases are patients ranging in age from years [20]. This is a descriptive study evaluating the performance of PCR-based methods for TB diagnosis and susceptibility to RMP and INH in stool samples from PTB patients compared to LJ culture for TB diagnosis and the conventional PM for susceptibility testing. Patients and specimens. Patients were diagnosed and treated by the Egyptian national tuberculosis program following the recommendations of the World Health Organization (WHO). The inclusion criteria for this study were patients between 10 and 45 years old with clinical and laboratory-proven PTB (either sputum microscopy and/or culture-positive for TB). The exclusion criteria was clinical symptoms or physical signs suggestive of extrapulmonary TB or any disease other than PTB, as determined by clinical examination (Table 1). Healthy individuals (control group) received the BCG vaccine and had no history or symptoms of TB. Pretreated patients represent a clinical model for newly diagnosed patients who have an increased risk of RMPand INH-resistant M.tb. Follow-up patients who are receiving treatment represent a clinical model for patients who have a poor response to treatment for one month and they are neither relapse patients nor have had TB treatment previously. Three successive stool and sputum specimens were collected in sterile receptacles at three-day intervals from each patient prior to treatment or as soon as possible after starting treatment for microbiological and molecular examinations of MTC organisms (Figure 1). Sputum Processing. Sputum samples were prepared using standard procedures for mycobacterial laboratories. The samples were digested and decontaminated with NALC-NaOH. A Ziehl-Neelsen smear was prepared and the presence of acid-fast bacilli was noted [21]. A

3 618 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 Figure 1. Study scheme showing the numbers of patients and samples in each group. sputum culture was performed on LJ medium for TB diagnosis and susceptibility testing for INH and RMP was performed using the PM on Middlebrook 7H11, according to the Clinical and Laboratory Standards Institute (CLSI) procedures and recommended critical concentrations [22]. Stool processing. Approximately 100 mg of stool sample was mixed with 6 ml of sterile distilled water and left standing for 15 min at room temperature. Then, 2 ml of the supernatant was decontaminated using the sodium hydroxide N-acetyl-L-cysteine technique [23]. DNA extraction. DNA was extracted from decontaminated stool samples using 5% Chelex -100 (Bio-Rad Laboratories, CA, USA) in sterile H2O as described previously [24]. Molecular diagnosis. PCR for the detection of M.tb. PCR-based detection of M.tb was performed via specific gene amplification using Genekam kits for M.tb identification (Genekam Biotechnology AG, Duisburg, Germany) following the manufacturer s instructions. The PCR products were subjected to electrophoresis and viewed using a UV light to detect a 317-bp band. The Genotype MTBDR-plus assay for INH and RMP susceptibility testing. The Genotype MTBDR-plus assay (HainLifescience, Nehren, Germany) was performed as recommended by the manufacturer. Hybridization and detection were performed in an automated washing and shaking device (Profiblot; Tekan, Maennedorf, Switzerland). Each strip contains 17 probes, including five amplification and hybridization controls to verify the test procedures, five rpob wildtype probes and four mutant probes, and one katg wild-type probe and two mutant probes. Data analysis. All procedures were performed in triplicate and the mean was determined for statistical analysis. Sensitivity and specificity were calculated for detection of resistance using a PCR-based method and the 95% confidence interval (CI) was also determined. Compared to the gold standard) dlj culture for PTB diagnosis and PM for RMP and INH susceptibility testing), positive and negative predicted values were ignored because the sample sizes in the positive (disease present) and the negative (disease absent) groups did not reflect the real prevalence of the disease. The data were analyzed with MedCalc software (MedCalc, Mariakerke, Belgium). Receiver operating characteristic (ROC) curve analysis was also performed using MedCalc.

4 Table 2. Results of PCR based method for detection of Mtb in stool samples compared to conventional LJ culture method. Patients No of samples Both Both PCR +ve PCR -ve Sensitivity% Specificity% AUC +ve -ve LJ-ve LJ +ve (95% CI) (95% CI) Newly diagnosed ( ) ( ) Follow up (83-100) 80 ( ) Control ND ND ND +ve: positive, ve: negative, PCR: PCR based method (Genekam), LJ: Sputum LJ culture, method AUC: Area Under ROC Curve. Table 3. Results of PCR based method for detection of Mtb in stool samples compared to conventional LJ culture method. Newly diagnosed patients No of Both S Both R PCR S PCR R Sensitivity% Specificity% AUC isolates PM R PM S (95% CI) (95% CI) INH ( ) 98.1 ( ) RMP ( ) 97.8 ( ) Follow up patients Direct Molecular Methods for Diagnosis of Pulmonary Tuberculosis 619 INH ( ) 94.4 ( ) RMP ( ) 94.4 ( ) S: sensitive, R: resistant, PCRS: PCR-based method, PM: conventional agar proportion method, AUC: area under ROC curve. Results In this study, stool samples from 100 PTB patients were analyzed using a PCR-based method (Genekam) for the detection of M.tb. The results of this molecular method was compared to sputum LJ culture as a reference method. Of the 100 patients tested, 94 were positive according to the molecular method, and all 20 control samples were negative. This data corresponds to a sensitivity of 100% and a specificity of 95.24% for newly diagnosed patients. The Genekam method also showed 100% sensitivity and 80% specificity for follow-up patients (samples from the same patient were considered as one diagnostic sample) compared to the sputum LJ culture method (Table 2). For all paired sputum and stool samples it was we found that results tend to be positive with increasing of number of acid-fast bacilli in film, and the incidence of false-positive results increased in patients that had received treatment. The 74 samples that showed positive results with the PCR-based method for M.tb detection were subjected to RMP and INH susceptibility testing using the Genotype MTBDR-plus assay. This method showed 86.4% and 100% sensitivity and 98.1% and 97.8% specificity for INH and RMP, respectively, in newly diagnosed patients compared to PM. For follow-up patients, the Genotype MTBDR-plus assay showed 85.7% sensitivity and 94.4% specificity for both INH and RMP compared to PM (Table 2). To assess the performance of the PCR-based method for M.tb detection and RMP and INH susceptibility testing in stool samples, ROC curve analysis was performed. The area under the curve (AUC) values was and for the detection of M.tb in samples from newly diagnosed and follow-up patients, respectively. For INH and RMP susceptibility testing in newly diagnosed patients, the AUC values were and for INH and RMP, respectively. In follow-up patients, the AUC values were and for INH and RMP, respectively (Table 2).

5 620 Annals of Clinical & Laboratory Science, vol. 46, no. 6, 2016 Discussion PTB diagnosis and susceptibility testing methods are either time consuming, in cases of culture-based methods, or require the use of radioisotopes, and can also be costly to perform [24]. The aim of this study was to evaluate PCR-based methods for the rapid detection of swallowed M.tb. These methods could potentially be used for diagnosis and drug susceptibility testing of patients with PTB. Stool samples are ideal for PTB diagnosis and susceptibility testing because they are obtained by non-invasive measures. Our findings showed that diagnosis of PTB via detection of M.tb in stool samples using the PCRbased Genekam kit had 100% sensitivity and 95.24% specificity for newly diagnosed patients and 100% sensitivity and 80% specificity for follow-up patients in comparison with the LJ culture as the gold standard for PTB diagnosis. To the best of our knowledge, there are no published studies that have evaluated the Genekam kit, but several groups have evaluated PCR-based methods using stool samples. These studies showed that clinical sensitivity of the PCR methods ranged from 0.74 to 1.00 [24-26]. Other studies report a relatively low PCR sensitivity, e.g., the studies by Pierre et al [28] and Soini et al [29]. These low sensitivities in PCR studies may be explained by suboptimal assay conditions [30]. Susceptibility test results using the Genotype MTBDR-plus assay showed 86.4% and 100% sensitivity and 98.1% and 97.8% specificity for INH and RMP, respectively, in newly diagnosed patients and 85.7% sensitivity and 94.4% specificity for both INH and RMP in follow-up patients compared to PM. Similar results were obtained by Doris et al. The Genotype MTBDR-plus assay demonstrated high sensitivity and specificity (99% and 100%, respectively) for detection of RMP resistance compared to the conventional DST [31]. In previous research, the sputum heteroduplex- PCR test had a 97% agreement with culture-based testing for the determination of rifampin resistance [18]. Although molecular-based methods are rapid and could be promising techniques for PTB diagnosis and susceptibility testing, there are several limitations. The detection of MTC depends on the presence of bacterial DNA in stool samples and by itself is not a diagnosis of active PTB because MTC organisms killed by anti-tuberculosis drugs can release DNA and lead to false-positive results. PCRbased PTB diagnosis should be performed on at least three samples from each patient to exclude the random absence of DNA or bacilli in clinical specimens that may lead to false-negative results. Furthermore, we found that PCR-based M.tb detection was affected by treatment duration via its effects on bacterial viability and subsequent falsepositive results; therefore, this tool should not be used for follow-up patients. Additionally, these molecular methods require special precautions and trained technicians. All of these limitations must be taken into account when PCR-based methods are used for diagnosis and susceptibility testing of PTB using stool samples, and these PCR-based methods cannot be used alone for PTB diagnosis and susceptibility testing. We conclude from this study that PCR-based methods for the detection and susceptibility testing of M.tb are promising, especially considering the ease of sample collection and the rapidity of diagnosis are taken into account. Despite the limitations described above, PCR-based methods could be powerful tools for the diagnosis of PTB and for susceptibility testing in patients who are unable to expectorate sputum. Acknowledgment This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center. References 1. World Health Organization (2014) Global Tuberculosis Control. World Health Organization, Geneva. 2. Pfyffer G. (2007). Mycobacterium: General Characteristics, Laboratory detection and Staining Procedures, 9th edn, pp Edited by Murray PR, Barron EJ, Jorgensen J H, Landry M L, Pfaller MA. Washington DC: American Society for Microbiology. 3. Oberhelman RA, Soto-Castellares G, Caviedes L, Castillo ME, Kissinger P, Moore DA, Evans C, Gilman RH (2006). Improved recovery of Mycobacterium tuberculosis from children using the microscopic observation drug susceptibility method. Pediatrics 2006; 118: Andresen D, Microbiological diagnostic procedures in respiratory infections: mycobacterial infections. Paediatr Respir Rev 2007;8: Owens S, Abdel-Rahman IE, Balyejusa S, Musoke P, Cooke RP, Parry CM, Coulter JB. Nasopharyngeal aspiration for diagnosis of pulmonary tuberculosis. Arch Dis Child 2007;92:

6 Direct Molecular Methods for Diagnosis of Pulmonary Tuberculosis Chierakul N, Anantasetagoon T, Chaiprasert A, Tingtoy N. Diagnostic value of gastric aspirate smear and polymerase chain reaction in smear-negative pulmonary tuberculosis. Respirology 2003;8: Ding H, MaY, Rao X, Jiao A, Liu X. The role of flexible bronchoscopy in pediatric pulmonary tuberculosis. J Trop Pediatr 2008;54: Vargas D, Garcia L, Gilman RH, Evans C, Ticona E, Navincopa M, Luo RF, Caviedes L, Hong C. Diagnosis of sputum-scarce HIV-associated pulmonary tuberculosis in Lima, Peru. Lancet 2005;365: Vandal OH, Pierini LM, Schnappinger D, Nathan CF, Ehrt S. A membrane protein preserves intrabacterial ph in intraphagosomal Mycobacterium tuberculosis. Nat Med 2008;14: Donald PR, Schaaf HS, Gie RP, Beyers N, Sirgel FA, Venter A. Stool microscopy and culture to assist the diagnosis of pulmonary tuberculosis in childhood. J Trop Pediatr 1996;42: Zhang Y and Telenti A. (2000). Genetics of drug resistance in Mycobacterium tuberculosis, p In G. F. Hatfull and Jacobs WR., Jr. (ed.), Molecular genetics of mycobacteria. ASM Press, Washington, D.C. 12. Kent PT and Kubica GP. (1985). Public health mycobacteriology. A guide for the level III laboratory. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Atlanta, GA, USA. 13. Roberts GD, Goodman NL, Heifets. Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens. J of Clin Microbiol 1983;18: Kox LF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van Leeuwen J, van Heusden S, Kuijper S and Kolk AH. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1994; 32: Lachnik J, Ackermann B, Bohrssen A, Maass S, Diephaus C, Puncken A, Stermann M, and Bange FC. Rapid-cycle PCR and fluorimetry for detection of mycobacteria. J Clin Microbiol 2002;40: Montenegro SH, Gilman RH, Sheen P, Cama R, Caviedes L, Hopper T, Chambers R, Oberhelman RA. Improved detection of Mycobacterium tuberculosis in Peruvian children by use of a heminested IS6110 polymerase chain reaction assay. Clin. Infect. Dis.2003; 36: Drobniewski FA, Wilson SM. The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis a molecular story. J Med Microbiol 1998;47: Mayta H, Gilman RH, Arenas F, Valencia T, Caviedes L, Montenegro SH, Ticona E, Ortiz J, Chumpitaz R, Evans CA, Williams DL. Evaluation of a PCR-based universal heteroduplex generator assay as a tool for rapid detection of multidrugresistant Mycobacterium tuberculosis in Peru. J Clin Microbiol 2003; 41: Moore DA, Evans CA, Gilman RH, Caviedes L, Coronel J, Vivar A, Sanchez E, Pinedo Y, Saravia JC, Salazar C, Oberhelman R, Hollm-Delgado MG, LaChira D, Escombe AR, Friedland JS. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med. 2006;355: El Din MAT, El Maraghy AA, Hay AHRA. Adverse reactions among patients being treated for multi-drug resistant tuberculosis at Abbassia Chest Hospital. Egyptian Journal of Chest Diseases and Tuberculosis. 2015; 64(4): Kawai V, Soto G, Gilman RH, Bautista CT, Caviedes L, Huaroto L, Ticona E, Ortiz J, Tovar M, Chavez V, Rodriguez R, Escombe AR, Evans CA. Tuberculosis mortality, drug resistance, and infectiousness in patients with and without HIV infection in Peru. Am J Trop Med Hyg 2006 ;75: Kent PT, Kubica GP Public health mycobacteriology: a guide for the level III laboratory. USDHHS, Centers for Disease Control, Atlanta, GA. 23. National Committee for Clinical Laboratory Standards: Susceptibility testing for Mycobacteria, Nocardiae, and other aerobic Actinomycetes; approved standard 2003;23: NCCLS document, pm-24a. NCCLS: Wayne, PA Chakravorty S, Tyagi JS. Novel use of guanidiniumisothiocyanate in the isolation of Mycobacterium tuberculosis DNA from clinical material. FEMS Microbiol Lett 2001;205: Hermans PM, Schuitema AJ, van Soolingen D, Verstynen CJ, Bik EM, Thole JR, Kolk AJ, van Embden J A. Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J ClinMicrobiol 1990;28: Forbes BA, Hicks K. Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction. J Clin Microbiol 1993; 31: Jonas V, Alden MJ, Curry JI. Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by amplification of rrna. J Clin Microbiol 1993; 31: Pierre C, Lecossier D, Boussougnant Y, Bocart D, Joly V, Veni P, Hance A. Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol 1991; 29: Soini H, Skurnik M, Liippo K, Tala E, Vilganen MK. Detection and identification of mycobacteria by amplification of a segment of the gene coding for the 32-kilodalton protein. J Clin Microbiol 1992;30: Beige, J., Lokies, J., Schaberg, T., Finckh, U., Fischer, M., Mauch, H.,Rolfs, A. (1995). Clinical evaluation of a Mycobacterium tuberculosis PCR assay. Journal of clinical microbiology, 33(1), Doris H, Michael W, Tanja K, Elvira R, Stefan N. Use of the Genotype MTBDR Assay for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates. J Clin Micrbiol 2005;43 :